Localization and characterization of the region encoding catabolism of mannopinic acid from the octopine-type Ti plasmid pTi15955

The region of the octopine-type tumor-inducing (Ti) plasmid pTi15955 encoding catabolism of mannopinic acid was localized to an 18-kilobase (kb) segment mapping between Ti plasmid coordinates 70 and 88. A subclone containing only this region normally did not allow utilization of any other mannityl opine. However, spontaneous mutants of the plasmid were isolated that conferred catabolism of agropinic acid. While the mutants remained regulated for mannopinic acid utilization, induction by this opine resulted in increased activity associated with agropinic acid catabolism. Respirometric studies and results from growth assays on mannopinic acid analogues indicated that the genes encoded on the 18-kb subclone were regulated in a wild-type fashion. By means of these analogues, spontaneous constitutive mutants were isolated. In several cases, the mutant phenotype was associated with an insertion event mapping within a 300-base pair region of the subclone insert. Although constitutive for catabolism of mannopinic acid, these mutants remained unable to catabolize any of the other mannityl opines. Segregation studies and genetic reconstitution experiments showed that one of the subclones isolated directly in Agrobacterium was actually composed of two complementing recombinant plasmids contained in the same cell. This indicated that the genes encoding mannopinic acid catabolism were organized into at least two complementation groups. These results point to a high degree of complexity in the organization and regulation of Ti plasmid genes encoding catabolism of a relatively simple carbon source.     

Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955

Summary The genetic organization of functions responsible for mannityl opine catabolism of the Ti plasmid of Agrobacterium tumefaciens strain 15955 was investigated. A partial HindIII digest of pTi15955 was cloned into a broad host range cosmid and the clones obtained were tested for ability to confer mannityl opine degradation upon Agrobacterium. Inserts containing genes for catabolism of mannopinic acid, mannopine, agropine, and agropinic acid were obtained, spanning a segment of 43 kb on the Ti plasmid. Two clones conferring upon Agrobacterium the ability to catabolize the mannityl opines were mobilized to several Rhizobium sp., to Pseudomonas putida and P. fluorescens and to Escherichia coli. The catabolic functions were phenotypically expressed in all Rhizobium sp. tested, and in P. fluorescens, but not in P. putida or in E. coli.     

Opine utilization by Agrobacterium spp.: octopine-type Ti plasmids encode two pathways for mannopinic acid degradation.

Octopine-type strains of Agrobacterium tumefaciens degrade the opine mannopinic acid through a specific pathway which involves cleavage of the molecule at the C--N bond between the amino acid and the sugar moieties. Mannose was identified as a product of the reaction. This pathway was inducible by mannopinic and agropinic acids, but not by mannopine or agropine, the two other mannityl opines. The transport system for this pathway appeared to be specific for mannopinic acid. A second, nonspecific pathway for mannopinic acid degradation was also identified. This involved some of the catabolic functions associated with the metabolism of mannopine and agropine. This second pathway was inducible by mannopine and agropine but not by mannopinic or agropinic acids. The transport system for this pathway appeared to have a broad specificity. Transposon Tn5 insertion mutants affected in the specific catabolic pathway were isolated and analyzed. These mutants continued to catabolize mannopine and agropine. Both mapped to a region of the Ti plasmid previously shown to be associated with the catabolism of mannopinic acid. Restriction enzyme analysis of the Ti plasmid from strain 89.10, an octopine strain that is naturally unable to utilize mannopinic acid, showed a deletion in this same region encoding the specific mannopinic acid degradation pathway. Analysis of recombinant clones showed that the second, nonspecific pathway was encoded in a region of the Ti plasmid associated with mannopine and agropine catabolism. This region shared no structural overlap with the segment of the plasmid encoding the specific mannopinic acid degradative pathway.     

Organization and regulation of the mannopine cyclase-associated opine catabolism genes in Agrobacterium tumefaciens 15955.

We have isolated and characterized Tn3HoHo1- and Tn5-induced mutants of a cosmid clone, pYDH208, which encodes the mannopine (MOP) cyclase-associated catabolism of MOP and agropine (AGR). Characterization of the transposon-induced lacZ fusion mutants by beta-galactosidase activity and mannityl opine utilization patterns identified at least 6 genetic units associated with the catabolism of these opines. Functions for the catabolism of MOP and mannopinic acid are encoded by a 16.4-kb region, whereas those for AGR are encoded by a 9.4-kb region located within the MOP catabolic locus. The induction pattern of catabolism shown by transposon insertion derivatives suggests that the catabolism of MOP, AGR, and mannopinic acid encoded by pYDH208 is regulated by at least two independent control elements. Kinetic uptake assays indicate that the clone encodes two transport systems for MOP and AGR, one constitutive and slow and the other inducible and rapid. Analysis of beta-galactosidase activities from lacZ reporter gene fusions indicated that expression of mannityl opine catabolic genes is not strongly repressed by sugars but is repressed by succinate when ammonium is the nitrogen source. The repression exerted by succinate was relieved when MOP was supplied as the sole source of nitrogen. This suggests that genes for opine catabolism encoded by pYDH208 are regulated, in part, by nitrogen availability.     

Functional role of the Ti plasmid-encoded catabolic mannopine cyclase in mannityl opine catabolism by Agrobacterium spp.

Catabolic mannopine (MOP) cyclase encoded by Ti or Ri plasmids lactonizes MOP to agropine (AGR). The gene of the octopine-type Ti plasmid pTi15955 encoding the catabolic MOP cyclase enzyme previously was localized to a 1.6-kb segment within a cosmid clone, pYDH208. A subclone containing only this region complemented the AGR catabolism-negative phenotype conferred by a derivative of the octopine-type plasmid pTiB6S3 containing a Tn7 insertion in the region encoding the MOP cyclase enzyme. Uptake assays of strains harboring pRiA4 or pArA4a, along with complementation analyses, indicate that MOP cyclase is not sufficient for catabolism of AGR but that the strains must also express an AGR transport system. To determine the requirement for MOP cyclase in opine catabolism unequivocally, a site-specific, nonpolar deletion mutation abolishing only MOP cyclase activity was introduced into pYDH208, a cosmid clone that confers utilization of MOP, AGR, and mannopinic acid (MOA). Strains harboring this MOP cyclase-negative mutant clone, pYDPH208, did not utilize AGR but continued to utilize MOP. Growth on AGR was restored in this strain upon introduction of clones encoding the pTi15955-derived catabolic or anabolic MOP cyclase genes. The induction pattern of MOA catabolism shown by strain NT1 harboring the MOP cyclase-deficient pYDPH208 suggests that AGR is converted into MOP by MOP cyclase and that MOP, but not AGR, induces catabolism of MOA. Genetic and biochemical analyses of MOP and AGR metabolism suggest that only the conversion of AGR to MOP is directly involved in catabolism of AGR, even though the reaction catalyzed by MOP cyclase predominantly lies in the lactonization of MOP to AGR.     

Mannityl opine analogs allow isolation of catabolic pathway regulatory mutants

Five virulent Agrobacterium spp. strains that can catabolize the mannityl opines mannopine (MOP), mannopinic acid ( MOA ), and agropinic acid (AGA) were tested for their ability to grow on analogs of these compounds. Analogs containing alternative amino acids replacing glutamic acid or glutamine were generally refused by these bacteria, but mutants were obtained that catabolized the entire family of analogs. In the case of strain C58C1 (pRi 8196), we demonstrated that typical mutants were constitutive for MOP uptake, whereas the wild-type parent was inducible by MOP. Analogs of MOA prepared from a variety of sugars instead of mannose were generally refused, except for a strain carrying pTi B6-806, which grew well on all such analogs. The analogs allowed selection of mutants of all strains. Although most wild-type strains were inducible for AGA uptake, typical mutants selected from strain C58C1 (pRi 8196) were found to be constitutive for uptake of AGA, as was the wild-type strain carrying pTi B6-806. Such constitutive mutants grew on all sugar analogs of MOP, MOA , and AGA tested. The pTi B6-806-containing strain was tested for growth on a more extended series of analogs, including tetrose , triose, diose , and disaccharide analogs, all of which were accepted. Only ketose analogs were refused. Selection of promiscuous regulatory mutants by the two types of opine analogs suggests that the repressor proteins of MOP and AGA permease/ catabolase systems are chiefly responsible for the specificity of the pathways.     

Mannopine and mannopinic acid as substrates for Arthrobacter sp. strain MBA209 and Pseudomonas putida NA513.

The characteristics of mannopine and mannopinic acid utilization by Agrobacterium tumefaciens B6S3, Arthrobacter sp. strain MBA209, and Pseudomonas putida NA513 were studied. Strain B6S3 utilized the four mannityl opines, mannopine, mannopinic acid, agropine, and agropinic acid. It also utilized several mannityl opine analogs, which were modified in either the sugar or the amino acid moiety. It utilized mannopine more rapidly after preincubation on mannopine, mannopinic acid, or glutamine than after pregrowth on glucose, mannose, or mannitol. Strains MBA209 and NA513 utilized mannopine and mannopinic acid, but not the other two mannityl opines. They utilized few mannityl opine analogs, sometimes because of failure to utilize the products of initial cleavage of the analog. Utilization of mannopine and mannopinic acid by strain NA513 was strictly dependent on prior growth on these substrates. A spontaneous regulatory variant of strain NA513 remained unable to utilize most of the mannityl opine analogs. Glutamine, mannose, and several analogs had no inhibitory effect on [14C]mannopine utilization by strain NA513.     

The arginine regulatory protein mediates repression by arginine of the operons encoding glutamate synthase and anabolic glutamate dehydrogenase in Pseudomonas aeruginosa

The arginine regulatory protein of Pseudomonas aeruginosa, ArgR, is essential for induction of operons that encode enzymes of the arginine succinyltransferase (AST) pathway, which is the primary route for arginine utilization by this organism under aerobic conditions. ArgR also induces the operon that encodes a catabolic NAD(+)-dependent glutamate dehydrogenase (GDH), which converts l-glutamate, the product of the AST pathway, in alpha-ketoglutarate. The studies reported here show that ArgR also participates in the regulation of other enzymes of glutamate metabolism. Exogenous arginine repressed the specific activities of glutamate synthase (GltBD) and anabolic NADP-dependent GDH (GdhA) in cell extracts of strain PAO1, and this repression was abolished in an argR mutant. The promoter regions of the gltBD operon, which encodes GltBD, and the gdhA gene, which encodes GdhA, were identified by primer extension experiments. Measurements of beta-galactosidase expression from gltB::lacZ and gdhA::lacZ translational fusions confirmed the role of ArgR in mediating arginine repression. Gel retardation assays demonstrated the binding of homogeneous ArgR to DNA fragments carrying the regulatory regions for the gltBD and gdhA genes. DNase I footprinting experiments showed that ArgR protects DNA sequences in the control regions for these genes that are homologous to the consensus sequence of the ArgR binding site. In silica analysis of genomic information for P. fluorescens, P. putida, and P. stutzeri suggests that the findings reported here regarding ArgR regulation of operons that encode enzymes of glutamate biosynthesis in P. aeruginosa likely apply to other pseudomonads.     

Mannityl opine accumulation and exudation by transgenic tobacco

Three genes from the T_R region of pTi15955 were introduced into tobacco (Nicotiana tabacum L.) to direct the synthesis of the mannityl opines from hexose sugars and glutamine or glutamate. Opines were present in all tissue types tested and accumulated to levels of 100 to 150 micrograms per milligram dry weight in root, stem, and leaf tissues. Opine-producing plants appeared normal with respect to morphology and development. Transgenic plants grown for 60 days under sterile autotrophic conditions produced up to 540 micrograms of the mannityl opines per milligrams dry weight of tissue as root exudates. Opines were also detected in leaf and seed washes from soil-grown plants.     

Organization of the agropine synthesis region of the T-DNA of the Ri plasmid from Agrobacterium rhizogenes.

The agropine/mannopine synthesis region of the TR region of the Ri plasmid of Agrobacterium rhizogenes strain A4 was localized on the basis of sequence similarity with probes from Ti plasmids of Agrobacterium tumefaciens and analysis of transposon insertions. The nucleotide sequence of the right part of the TR-DNA of pRiA4, encompassing the three genes involved in mannityl-opine synthesis, was determined and compared to the sequence of the corresponding region of the octopine-type Ti plasmid pTi15955. The organization of this region is strongly conserved between Ri and Ti plasmids, but the similarity is restricted to the coding sequences: no homology was detected in the 5' and 3' flanking sequences. The mas1' and ags proteins are the most conserved, showing more than 68% amino acid conservation, whereas the mas2' proteins are only 59% identical. Significant G/C content and codon usage differences are observed between pTi15955 and pRiA4. An open reading frame strongly similar to that of bacterial repressors is situated immediately to the right of the TR region.     

Site-specific mutagenesis in the TR-DNA region of octopine-type Ti plasmids

Site-specific insertion and deletion mutations affecting all six of the eukaryotic-like genes in the TR-DNA region of the octopine-type Ti plasmids pTil5955 or pTiA6 have been generated. None of the mutations affected virulence or tumor morphology on sunflower. Mutations in the coding regions of two of the genes resulted in tumors without any detectable mannopine, mannopinic acid or agropine, and mutations in either the coding region or in the 3′ untranslated region of a third gene eliminated biosynthesis of agropine, but not mannopine or mannopinic acid. Detection of two previously unobserved silver nitrate-positive substance in tumors incited by one of the mutant strains, together with data on the presence of opines in tumors incited by coinoculation with mixtures of different mutant strains, allowed us to propose the functional order of all three genes involved in the biosynthesis of mannopine, mannopinic acid and agropine. TR-DNA was absent in tumors incited by anAgrobacterium tumefaciens strain harboring a Ti plasmid in which the right border of the TR-DNA region was deleted.     

p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate.

Pseudomonas putida F1 utilizes p-cymene (p-isopropyltoluene) by an 11-step pathway through p-cumate (p-isopropylbenzoate) to isobutyrate, pyruvate, and acetyl coenzyme A. The cym operon, encoding the conversion of p-cymene to p-cumate, is located just upstream of the cmt operon, which encodes the further catabolism of p-cumate and is located, in turn, upstream of the tod (toluene catabolism) operon in P. putida F1. The sequences of an 11,236-bp DNA segment carrying the cym operon and a 915-bp DNA segment completing the sequence of the 2,673-bp DNA segment separating the cmt and tod operons have been determined and are discussed here. The cym operon contains six genes in the order cymBCAaAbDE. The gene products have been identified both by functional assays and by comparing deduced amino acid sequences to published sequences. Thus, cymAa and cymAb encode the two components of p-cymene monooxygenase, a hydroxylase and a reductase, respectively; cymB encodes p-cumic alcohol dehydrogenase; cymC encodes p-cumic aldehyde dehydrogenase; cymD encodes a putative outer membrane protein related to gene products of other aromatic hydrocarbon catabolic operons, but having an unknown function in p-cymene catabolism; and cymE encodes an acetyl coenzyme A synthetase whose role in this pathway is also unknown. Upstream of the cym operon is a regulatory gene, cymR. By using recombinant bacteria carrying either the operator-promoter region of the cym operon or the cmt operon upstream of genes encoding readily assayed enzymes, in the presence or absence of cymR, it was demonstrated that cymR encodes a repressor which controls expression of both the cym and cmt operons and is inducible by p-cumate but not p-cymene. Short (less than 350 bp) homologous DNA segments that are located upstream of cymR and between the cmt and tod operons may have been involved in recombination events that led to the current arrangement of cym, cmt, and tod genes in P. putida F1.     

Regulation of sialic acid transport and catabolism in Haemophilus influenzae

Virulence of nontypeable Haemophilus influenzae (NTHi) is dependent on the decoration of lipooligosaccharide with sialic acid. This sugar must be derived from the host, as NTHi cannot synthesize sialic acids. NTHi can also use sialic acid as a carbon source. The genes encoding the sialic acid transporter and the genes encoding the catabolic activities are localized to two divergently transcribed operons, the siaPT operon and the nan operon respectively. In this study, we identified SiaR as a repressor of sialic acid transport and catabolism in NTHi. Inactivation of siaR resulted in the unregulated expression of the genes in both operons. Unregulated catabolism of sialic acid in the siaR mutant resulted in the reduction of surface sialylation and an increase in serum sensitivity. In addition to SiaR-mediated repression, CRP, the cAMP receptor protein, was shown to activate expression of the siaPT operon but not the nan operon. We describe a model in which SiaR and CRP work to modulate intracellular sialic acid levels. Our results demonstrate the importance of SiaR-mediated regulation to balance the requirement of surface sialylation and the toxic accumulation of intracellular sialic acid.     

Molecular characterization of the oligopeptide permease of Salmonella typhimurium

The oligopeptide permease (Opp) of Salmonella typhimurium is a periplasmic binding protein-dependent transport system and handles any peptides containing from two to five amino acid residues. Opp plays an important nutritional role and is also required for the recycling of cell wall peptides. We have determined the nucleotide sequence of the opp operon. In addition to the four opp genes identified previously by genetic means (oppABCD) a fifth gene, oppF, is shown to be cotranscribed as part of the opp operon. Using reverse genetics, we show that oppF also encodes an essential component of the Opp transport system. The five proteins, OppABCDF, are shown to be the only proteins required for Opp function. Regulation of opp expression and of the differential expression of genes within the operon is investigated. We have devised a simple means of constructing lacZ gene fusions to any S. typhimurium chromosomal gene in vivo, using derivatives of bacteriophage Mu. Using this procedure, opp-lacZ gene fusions were selected. The resultant Opp-LacZ hybrid proteins were used to show that OppB, OppC and OppD are membrane-associated proteins. A detailed comparison of the Opp components with those of other binding protein-dependent transport systems provides insight into the mechanisms and evolution of these transport systems.